| Avian influenza(AI) is the syndrome of infected poultry caused by influenza A viruses. Haemagglutinin and neuraminidase are two most important surface antigens of AIV. According to their difference, influenza viruses are divided into16haemagglutinin (H1-16) and9neuraminidase (N1-9) subtypes. H9N2is a main kind of low pathogenic avian influenza. It widely spreads around the world. It shows the character that enzootic, lasting damage and hard to control. Therefore, it is important to make the right diagnosis and determine the subtype.Although the countries’strategies on avian influenza prevention and control are not the same, the rapid diagnosis and monitoring of avian influenza are the first step. In this study, two strain of monoclonal antibodies agaist H9-HA were used to establish two kind methods, Colloidal gold immunochromatograohic assay and sandwich ELISA for specific detection H9AIV.1Establishment of a double antibodies sandwich ELISA for detection of H9AIVIn this part, several strains of monoclonal antibodies agaist H9AIV were used. And a cross matching test was conducted after the two monoclonal antibodies were labeled with HRP. The results showed that H9-HA-1C3as coating antigen and H9-HA-2G4as testing antigen could give best results. The condition of working concentration of the ELISA was optimized. The best working concentration of H9-HA-1C3and H9-HA-2G4were1.25μg/mL and0.25μg/mL respectively. This method was specific for H9AIV, and has no cross reaction with Newcastle Disease Virus, Infectious Bronchitis Virus and other viruses. Compared with HA, the sensitivity of the ELISA could detect1/4HA titer of H9AIV.2Development of a test strip for rapid detection of H9AIVIn this part, nanometer colloidal gold was prepared by reducing gold chloride with sodium citrate, than30nm colloidal gold was acquired. The colloidal gold strip was developed according to the principle of double antibodies sandwich. Gold-labeled antibody H9-HA-1C3was dispensed onto the conjugate pad, the H9-HA-2G4monoclonal antibody was dispensed on the bottom of the NC membrane as the test line, while the rabbit anti-mouse IgG was dispensed at the upper position as the control line. When the samples contain H9antigen, two red bands will be visibled, otherwise, just a single band will be shown on the control line. The test strip could detect H9AIV specifically, and had no cross reaction with Newcastle Disease Virus, Infectious Bronchitis Virus and other viruses. The sensitivity of the test strip is1/4HA titer of H9AIV. The repeatability of different batches were the same batch. The sensitivity and specificity of the strip had no change even they were stored in4℃for6months.3Comparison of the two methods and preliminary applicationCompared the two methods in this research with the classical method, identification of the virus,we detected different samples with these mathods. We found the positive coincidence rate of H9ELISA with virus isolation was80%, while the negative coincidence rate was100%for tissue samples. The positive coincidence rate of H9test strip with virus isolation was70%, while the negative coincidence rate was100%. For throat swab samples collected from poultry free-market, the coincidence rate of H9test strip and H9ELISA, conpare with virus isolation, were97.9%.The negative coincidence rate for both method was100%. The specificities of the two H9AIV detection methods were good and no false-negative. The methods would be pratical value for application. |
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